Illuminating Dynamic Receptor Clustering in the Epidermal Growth Factor Receptor Signal Transduction Pathway Using Plasmon Coupling

摘要-促进健康相关研究的多样性的研究补充 EGFR簇的组织结构在控制和调节细胞增殖中起重要作用。 但这种空间控制机制的许多方面仍然存在 不够理解。监测活细胞中EGFR簇的结构和动力学的能力， 光学显微镜将有很长的路要走，以解开空间集群介导的机制， EGFR信号的调控。过去的研究旨在探测EGFT聚类光学涉及使用 量子点、荧光染料和金纳米颗粒。然而，这些标签中的每一个都有自己的挑战 和局限性。量子点闪烁具有细胞毒性，荧光染料漂白剂和金属纳米颗粒（NPs） 需要相对大的尺寸（> 40 nm）以在常规暗场或全内反射中可检测 显微镜虽然金纳米颗粒具有极端的光稳定性，但用这种大小的颗粒标记蛋白质可以 扰乱分子的结构和功能。所有这些挑战使得研究真正的 膜蛋白的动力学与常规技术。为了解决这些问题，我们建议 在这里开发一种干涉散射显微镜（iSCAT）来研究EGFR聚集的动力学， 以及EGFR簇通过NP标记的距离依赖性等离子体偶联信号的稳定性 EGFR对干涉检测方法仍然可以利用独特的物理特性 贵金属纳米颗粒（这些材料不闪烁或漂白），但与更小的纳米颗粒尺寸兼容。我们 将使用金和银NP标记，直径小至5 nm。 NP之间的距离依赖性等离子体偶联将用于监测NP标记的等离子体的缔合。 EGFR单体转化为二聚体和更大的寡聚体，以及它们在空间上结合成簇。自缔合 将通过NP标记物的等离子体共振的光谱位移来指示蛋白质的变化，其可以是 在远场检测和量化。

Summary – Research Supplement to Promote Diversity in Health-Related Research The organization of EGFR into clusters has shown to play an essential role in controlling and modulating he nature and intensity of cellular signaling but many aspects of this spatial control mechanism remain insufficiently understood. The ability to monitor the structure and dynamics of EGFR clusters in living cells with optical microscopies would go a long way to unravel the mechanisms underlying spatial clustering mediated control of EGFR signaling. Past studies aimed at probing EGFT clustering optically involved the use of quantum dots, fluorescent dyes, and gold nanoparticles. Each of these labels has, however, its own challenges and limitations. Quantum dots blink are cytotoxic, fluorescent dyes bleach and metal nanoparticles (NPs) require relatively large sizes (>40nm) to be detectable in conventional darkfield or total internal reflection microscopy. Although gold NPs have extreme photostabilities, labelling proteins with particle of this size can perturb the structure and function of the molecule. All these challenges make it difficult to study the true dynamics of the membrane protein with conventional techniques. To overcome these problems, we propose here to develop an interferometric scattering microscopy (iSCAT) to study the kinetics of EGFR clustering as well as the stability of EGFR clusters through distance-dependent plasmon coupling signals of NP-labelled EGFR. The interferometric detection approach can still take advantage of the unique photophysical properties of noble metal NPs (these materials don’t blink or bleach) but is compatible with much smaller NP sizes. We will use gold and silver NP labels with diameters as small as 5 nm in this project. Distance-dependent plasmon coupling between the NPs will be used to monitor the association of NP-labelled EGFR monomers into dimers and larger oligomers and their spatial association into clusters. The self-association of the proteins will be indicated by spectral shifts of the plasmon resonance of the NP labels, which can be detected and quantified in the far-field.